Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). Only DCs take up antigens at the site of infection and migrate to the lymph node where they present fragments of these antigens to naive T cells. The T cell receives the antigenic signal (signal 1: MHC-peptide/TCR), which causes the upregulation of the co-stimulatory molecule CD154. CD154, in turn, engages CD40 on the DC, thus activating the expression of other costimulatory molecules and cytokines. In the second phase, the T cell (for example via CD28) receives the costimulatory signal, which drives it to enter the cell cycle and become fully active. The CD154-CD40 interactions are also critical in B cell activation and immunoglobulin (Ig) production, and macrophage activation. There are clear indications that DCs are the first CD4+ cells that are infected during HIV and SIV infection through mucosal surfaces, and that infection of DCs leads to dysregulation of the signals delivered to and by T cells via CD154. In asymptomatic HIV infection, fewer DCs with reduced capacity to stimulate CD4+ T cell proliferation are found before evidence of T cell abnormalities, and these early changes in APCs may result in a decline in the production of CD4 memory T cells. In addition, AIDS patients and individuals with genetic defects in CD154 suffer from similar opportunistic infections. This proposal will study mechanisms that affect cellular immune responses to SIV infection. Specifically, we will study the role of the costimulatory molecule CD154 during infection of rhesus macaques with pathogenic virus (SIVmac251) or with a recombinant live-attenuated SIV that expresses rhesus CD154 (SIVHyCD154). We have cloned the CD154 gene from rhesus macaques and found that only 2 out 218 aminoacids in the extracellular region are different from the human protein. We have previously prepared replication-competent SIVs that are attenuated in vivo and express cytokines such as IFN-gamma and IL-18. We hypothesize that signaling by CD154 is disregulated during SIV infection, and that expression of CD154 by SIVHyCD154-infected cells may restore the activation pathway for T cell-dependent antigens: 1) SIVHyCD154-infected DCs will engage CD154 and CD40 at the cell surface and upregulate other costimulatory molecules; 2) SIVHyCD154-infected CD4+ T cells will engage their CD154 with CD40 on B cells, leading to B cell activation and expansion; and 3) SIVHyCD154-infected macrophages will engage CD154 and CD40 at the cell surface, leading to activation, release cytokines and NO, and APC function. Results from these experiments may lead to improvements in HIV vaccine design in at least two ways: i) better understanding of the mechanisms of immune dysregulation during HIV infection, or ii) prospect for safer live-attenuated vaccines.